Epidemiologic and clinical evidence has demonstrated that nonsteroidal anti-inflammatory drugs (NSAIDs), especially aspirin, protects against colorectal cancer (CRC) incidence and mortality. Elucidating the mechanism(s) responsible for the protective effect of NSAIDs could lead to major breakthroughs in the field of cancer chemoprevention and treatment. The most compelling evidence to date indicates that reduction of pro- inflammatory prostaglandin E2 (PGE2) production via inhibition of the cyclooxygenase pathway is responsible for part of this protective effect. Given that PGE2 can mediate the tumor-promoting effects of COX-1 and COX- 2, more selective pharmacological inhibition of PGE2 signaling may be efficacious in preventing CRC with avoiding the cardiovascular and other side effects associated with NSAID use. Although our group and others have extensively investigated the mechanisms by which PGE2 promotes CRC progression, it is still not fully understood how PGE2 accelerates CRC formation and progression. During the current funding period, our progress has led to many milestones in understanding the central role of PGE2 signaling in CRC and also provides the foundation for future investigation. Our preliminary data shows that PGE2 stimulates the expansion of colonic cancer stem cell (tumor-initiating cell) in vitro and in vivo. However, it is unclear whether PGE2 promotes CRC initiation and progression through enhancing cancer stem cell growth, maturation, and survival. We will address these questions in the Aim 1. Moreover, our preliminary results indicate that the CXCR2, one of downstream targets of PGE2, promotes chronic intestinal inflammation and colitis-associated tumorigenesis. We further provide the first evidence showing that CXCR2 is required to recruit myeloid-derived suppressor cells (MDSCs) to the colonic mucosal tissue. Previously, this receptor was thought to only mediate neutrophil migration to inflammatory sites. Although evidence for MDSC promotion of immunosuppression is accumulating, it remains unclear whether CXCR2-expressing MDSCs play a key role in connecting colonic chronic inflammation to colitis-associated carcinogenesis. Aim 2 is designed to address these questions. Finally, our preliminary data revealed that adoptive transfer of human natural killer (NK) cells significantly inhibited liver metastases in a mouse model of CRC metastasis. However, the role of NK cells in inhibiting metastatic CRC and preventing tumor recurrence after surgery remains unclear. In addition, we will evaluate whether inhibition of MDSC recruitment by CXCR2 antagonists and reduction of MDSC numbers in tumor by aspirin enhance the ability of NK cells to eliminate CRC liver metastases. We will address these issue in Aim 3. Collectively, the results from our renewal proposal will not only reveal comprehensive insights of how PGE2, CXCR2, MDSCs, and NK cells coordinately contribute to CRC initiation, progression, and metastasis, but also provide a rational for applying adoptive transfer of allogeneic NK cells with subverting tumor-induced immunosuppression as novel therapeutic approaches in metastatic and adjuvant therapies.